Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in K m and V max . In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37°C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.Epoxide hydrolases (EC 3.3.2.3) metabolize exogenous and endogenous epoxides by hydrolyzing them to vicinal diols, which are usually less reactive and less mutagenic because of their higher hydrophilicity. sEH is one of five epoxide hydrolases (the others are hepoxilin EH, leukotriene A 4 hydrolase, cholesterol EH, and microsomal EH), which differ in molecular weight, subcellular localization, pI and substrate specificity.Previous work suggests the existence of one hsEH gene localized to chromosomal region 8p21-p12 (Larsson et al., 1995). The human sEH gene (EPHX2) consists of 19 exons encoding 555 amino acids (Sandberg and Meijer, 1996). Because the human and mouse proteins are 73% identical (Beetham et al., 1995) with 100% identity in residues forming the catalytic triad, the crystal structure of murine sEH (Argiriadi et al., 1999) is a good model for predicting structurefunction correlations of the hsEH. Each monomer of the homodimeric mouse sEH has two domains: an N-terminal domain and a C-terminal catalytic domain connected by a proline rich linker (Argiriadi et al., 1999). The catalytic mechanism involves formation of a covalent alkylenzyme ester intermediate as a result of nucleophilic attack by Asp333. This is subsequently hydrolyzed with assistance of the general base His523 in a charge relay with...

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in α-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth–enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in α-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031Ϫ4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309Ϫc-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309Ϫc-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.Keywords : Saccharomyces cerevisiae; yeast; mitochondria; translation ; RNA processing.A novel aspect of mitochondrial gene expression is the con-[1, 4], may substitute for the function performed by IF3 in other prokaryotic-like translation systems. trol of mitochondrial RNA translation by gene-specific factors encoded by nuclear genes (for review, see [1Ϫ3]). In SaccharoMitochondrial RNA processing is also controlled by genespecific factors encoded by nuclear genes (for review, see [2, myces cerevisiae, it is likely that each mitochondrial mRNA employs specific factors required for translation initiation. These 3]). Several mitochondrial genes in S. cerevisiae contain introns may act in addition to general translation initiation factors, or which are thought to be spliced by ribozyme mechanisms faciliinstead of them. A nuclear gene for mitochondrial translation tated by protein factors (RNA chaperones) that facilitate proper initiation factor 2 (IF2) has been found, but a clear homologue folding of the intron ribozymes. Both mitochondrial-encoded of prokaryotic translation initiation factor 3 (IF3) has not been maturases and nuclear-encoded proteins required for mitofound, despite the availability of the complete DNA sequence chondrial RNA splicing have been identified. Additionally, of the S. cerevisiae genome. The function of yeast gene-specific nuclear-encoded trans-acting proteins required for mitochondrial mitochondrial translation factors such as Pet122p or Cbs2p, RNA 5′ or 3′ end processing or mRNA stability...

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of A and β subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinylCoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase A subunit, and this band was absent in ∆lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.Keywords : succinyl-CoA ligase ; tricarboxylic acid cycle; mitochondria; Saccharomyces cerevisiae; yeast.Succinyl-CoA ligase (succinyl-CoA synthetase ; succinate thiokinase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate [1]. This is the only enzyme of the tricarboxylic acid (TCA) cycle that directly produces a highenergy phosphate. Succinyl-CoA ligase is composed of A and β subunits that function either as dimers or tetramers. The Escherichia coli enzyme is an A 2 β 2 tetramer, in which two independently active heterodimers are capable of synergistic interaction with the substrate [1Ϫ3]. The porcine heart mitochondrial enzyme has also been well characterized and, like other eukaryotic isoforms, it functions as an Aβ heterodimer [4]. In both prokaryotes and eukaryotes, the A subunit is phosphorylated on a conserved histidine residue, and the phosphate is subsequently transferred to form ATP or GTP [1]. Alteration of this residue by mutagenesis results in an inactive enzyme [5].Isoforms of succinyl-CoA ligase have been distinguished on the basis of their nucleotide preference for either GTP or ATP. The E. coli and Pseudomonas aeruginosa enzymes can utilize either ATP or GTP [1,6,7]. Eukaryotic succinyl-CoA ligases can be either ATP or GTP dependent [1]. While the ATP-dependent form is involved in TCA-cycle metabolism, the GTP-dependent form has been associated with ketone-body breakdown and heme biosynthesis [8,9]. The purified enzyme from Dichtyostelium discoideum shows specificity for GTP, while the enzyme Correspondence to M. T. McCammon,